ε‐poly‐L‐lysine‐modified polydopamine nanoparticles for targeted photothermal therapy of drug‐resistant bacterial keratitis

Abstract Bacterial keratitis can lead to intraocular infection and even blindness without prompt and potent treatments. Currently, clinical abuse of antibiotics encouraged the evolution of resistant bacteria. Conventional antibiotic eye drops based keratitis treatment has been heavily restricted due to the lack of bactericidal efficiency and easy induction of bacterial resistance. Hence, developing an effective treatment strategy for bacterial keratitis is of great significance. In this work, we investigated ε‐poly‐l‐lysine (EPL)‐modified polydopamine (PDA) nanoparticles (EPL@PDA NPs)‐mediated antibacterial photothermal therapy (aPTT), to cope with methicillin‐resistant Staphylococcus aureus (MRSA)‐induced keratitis. The surface modification of cationic peptide EPL enables EPL@PDA NPs to specifically target negatively charged MRSA and induces local hyperthermia to kill the bacteria under low ambient temperature. Under near‐infrared (NIR) irradiation, the sterilization efficiency of EPL@PDA NPs suspension for MRSA in vitro was up to 99.96%. The EPL@PDA‐mediated aPTT presented potent antibacterial efficacy in treating MRSA‐induced keratitis with little corneal epithelial cytotoxicity and good biocompatibility. In conclusion, the bacterial‐targeting aPTT platform in this work provides a prospective method for the management of MRSA‐induced refractory bacterial keratitis.


| INTRODUCTION
As a purulent corneal infection caused by bacteria, bacterial keratitis can lead to corneal melting, scarring, and perforation if not treated promptly and potently. 1,2 Conventional clinical protocol includes the topical administration of broad-spectrum antibiotic eye drops.
Whereas the development of novel antibiotics has encountered huge obstacles to keep pace with the evolution of multidrug-resistant (MDR) bacteria. 3,4 Selection of sensitive antibiotics requires timeconsuming laboratory tests, which could delay the illness course.
Moreover, the toxicity caused by the high dose of antibiotics also needs to be taken into consideration. 5,6 To address these challenges, novel board-spectrum therapeutic approaches such as metal nanoparticles, nanozymes, cationic species, photothermal therapy (PTT), and photodynamic therapy (PDT) have been investigated. [7][8][9][10][11][12][13][14][15] PTT utilizes photothermal transduction agents' (PTAs) photothermal conversion effect to generate heat energy under external light irradiation, and raise the surrounding environment's temperature, to eliminate the target cancer cells, bacteria, and other pathogenic microorganisms. [16][17][18][19][20] The broad-spectrum bactericidal efficiency and relatively straightforward sterilization process of PTT have aroused enormous attention. PTT has been widely reported to exhibit excellent performance in managing diseases such as cancer and bacterial infections currently. [21][22][23][24][25][26] Radiated PTAs can locally raise the temperature (over 50 C) to cause microbial death by induction of oxidative stress, leading to protein hyperthermia-denaturation, nucleic acid degradation, and cell membrane destruction. 27,28 Hence, such physical therapy has a huge natural strength in dealing with bacterial infections caused by MDR strains, such as ampicillin-resistant Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA). 8,[29][30][31] Among a variety of PTAs, polydopamine (PDA) has distinguished itself owing to excellent photothermal conversion efficiency and brilliant biocompatibility compared to traditional metallic nanostructures. 32 Its photothermal conversion efficiency can reach 40%, which promotes its application in the management of cancer and bacterial infection. 33,34 Furthermore, PDA's catechol structure is easy to be oxidized to diquinone structure, so it can react with various functional groups (e.g. amine and thiol) via Michael addition and/or Schiff base reaction, and easily be surface modified. 35,36 Additionally, the preparation of PDA is rather straightforward and requires mild conditions, as dopamine can self-polymerize to form PDA in a weak alkaline reaction environment. 37 However, conventional aPTT lacks the specific bactericidal ability, and the hyperthermia produced by aPTT will affect and damage the surrounding tissues during sterilization. To diminish unnecessary tissue lesions and enhance bactericidal efficiency, specific modification of PTAs aiming at improving their targeting ability and concentrating the heat on bacteria has become a feasible strategy. 35,[38][39][40] ε-poly-L-lysine (EPL) is a natural cationic homopolymer containing 25-35 L-lysine residues. EPL can selectively attach to bacteria surfaces in the physiological environment by electrostatic interaction with the negatively charged cytoplasmic membrane. 41,42 Moreover, EPL is biodegradable, nontoxic, and more affordable to produce than other cationic peptides. 43 In this study, EPL-modified PDA composite nanoparticles (EPL@PDA NPs) based aPTT was developed to achieve low-temperature sterilization in the management of MDR MRSA-induced keratitis (Scheme 1). As a cationic peptide, EPL is capable of binding to negatively charged bacterial cytoplasmic membrane by electrostatic interaction in nature. 44,45 The surface modification of EPL enables EPL@PDA NPs to specifically target negatively charged bacteria. Under the irradiation of NIR lasers (808 nm), EPL@PDA NPs can generate local hyperthermia on the surface of MRSA. The concentration of free EPL@PDA NPs decreased significantly, as they were enriched around bacteria. Therefore, the aPTT platform can efficiently kill the bacteria under low ambient temperature, which avoids thermal damage to surrounding tissues to a large extent. Hence, EPL@PDA NPs mediated aPTT can achieve rapid and efficient eradication of bacteria, providing a novel and prospective method for treating MRSA-caused corneal infection.

| Photothermal conversion performance of EPL@PDA NPs
PDA NPs display absorption in the NIR region (760-900 nm) ( Figure 2a). In this work, NIR laser (808 nm) was exploited to investigate EPL@PDA NPs' photothermal conversion capability. We explored the relationship between laser intensity, concentration, irradiation time, and temperature rising degree. Specifically, EPL@PDA NPs in PBS were irradiated by 808 nm NIR light for 10 min, respectively. The photothermal activity of EPL@PDA NPs (concentration from 50 to 300 μg mL À1 ) was investigated by comparing the temperature change when exposed to NIR light

| Selective adhesion of EPL@PDA NPs on bacterial membrane
It is well known that PDA NPs-based aPTT displayed satisfactory bactericidal effect. However, the general temperature rise may cause unwelcome damage to affected tissues. Developing bacteria-targeting PTAs to achieve sterilization under a low temperature could improve therapeutic efficiency and reduce side effects. 18 As EPL@PDA NPs were fabricated to be positively charged, they were inclined to adhere to the bacteria. SEM was utilized to observe and characterize the selective adhesion between EPL@PDA NPs and MRSA. After PBS, PDA and EPL@PDA NPs (200 μg mL À1 , 100 μL) were incubated with 100 μL 5 Â 10 7 colony forming units ml À1 (CFU mL À1 ) MRSA suspension for 1 h, respectively, the mixed suspensions were either exposed to 808 nm NIR laser (3.64 W cm À2 , 10 min) or not. The quantity of EPL@PDA NPs adhered to MRSA significantly exceeded PDA NPs, suggesting a strong bacterial targeting activity of EPL@PDA NPs

| In vitro bactericidal performance to MDR bacteria
The antibacterial performance of EPL@PDA NPs mediated aPTT against MRSA was investigated via the typical spread plate method and were incubated for 1 h, respectively. NIR irradiation groups were exposed to 808 nm NIR laser (3.64 W cm À2 , 10 min). When PBS was incubated with MRSA with or without irradiation, the CFU of bacteria Under NIR irradiation, the temperature of MRSA surface to which EPL@PDA NPs attached can be estimated to reach 100 C before the heat balance. 33 Such local high temperature can induce a significant bactericidal effect, and the sterilization rate was up to 99.61% (200 μg mL À1 ) and 99.96% (300 μg mL À1 ) while the general temperature of the suspension remained at a relatively low level ($49 C and MRSA was mixed with EPL@PDA NPs and exposed to NIR, they displayed the strongest PI fluorescence. Hence, we believe that the modification of EPL significantly enhanced the sterilization efficiency of aPTT.

| In vivo anti-infective performance on MRSAinfected bacterial keratitis model
Previous results have proved the brilliant antibacterial ability of EPL@PDA NPs based aPTT platform, which inspired us to further explore its clinical transformation potential for the management of intractable bacterial keratitis. Therefore, we investigated its therapeutic efficacy on a murine MRSA-induced keratitis model. After the keratitis model was established, an oval focal abscess with a gray-whiteish corneal stroma infiltration with obvious boundaries could be observed and recorded via slit lamp (Figure 4c). Each group was given the corresponding treatment. During the aPTT process, the local temperature rose to 41.5 C (PDA) and 41.8 C (EPL@PDA) within the cornea (Figure 4a,b). Owing to the bacterial target effect of EPL@PDA NPs, we were able to conduct the aPTT progress even under such a low temperature without compromising therapeutic efficiency.
The evaluation of therapeutic effect was mainly based on the area and depth of abscess, specifically manifested as the scope and transparency of cornea opaque. 46 Due to the heavy bacteria burden, the

| Biotoxicity evaluation
The biocompatibility of materials directly determines whether clinical translation can be carried out. 47 Human corneal epithelial cells  (Figure 5k-o). H&E staining was conducted on tissue sections of the main murine organs, namely the heart, liver, kidney, lung, and spleen (Figure 5p). Negligible damage was observed in the tissue morphology of the mouse organs. The excellent biocompatibility of the aPTT platform is mainly owing to the low toxicity of EPL@PDA.
Besides, low dosing frequency diminishes the amount of the drug circulating throughout the body, which further reduces drug-induced tissue damage. Therefore, the EPL@PDA-based aPTT platform displays low biotoxicity, indicating its potential for clinical transformation in managing bacterial keratitis.

| CONCLUSION
In summary, EPL@PDA NPs were fabricated to conduct a targeted aPTT-mediated antibacterial process and effectively manage MRSAinduced keratitis under low ambient temperature. In this platform, the negative charge of PDA NPs was reversed by EPL modification.     In brief, MRSA (1 μL, 1 Â 10 7 CFU mL À1 ) was injected into the murine corneal stroma. After 16 h of inoculation, the murine cornea was observed using a slit lamp, and the emergence of opacity and edema prompts a successful establishment of the bacterial keratitis model (Day 0). Infected mice were randomly divided into four groups and treated with different regimens, namely Ctrl (PBS), PDA (PDA NPs, 300 μg mL À1 , NIR), EPL (EPL, 1%), and EPL@PDA (EPL@PDA NPs PBS solution, 300 μg mL À1 , NIR). In the PTT process, PDA or EPL@PDA NPs suspension was dropped on the ocular surface three times (once every 2 min). Then, irradiate the eye for 5 min using 808 nm (2.55 W cm À2 ) laser once. Subsequently, the progression of the keratitis is observed using slit lamp and recorded by camera, and the mice were sacrificed on Day 10. Then, harvest the eyeballs for corneal homogenate plate counting, histological morphology observation, and extract the main organs (namely heart, liver, kidney, lung, and spleen) to observe the toxicity that treatment regimens exert on mice.

DATA AVAILABILITY STATEMENT
Research data are not shared.